Biology subjects

Essen, L.-O.

Publications and source records attributed to Essen, L.-O..

7 recordsLinked to original sources

bioRxiv2026

Amino acid Mutations at the Interface of Sudan Virus VP40 alter transport and budding

The matrix protein VP40 of orthoebolaviruses coordinates virion release and downregulates viral RNA synthesis through distinct oligomeric states, including dimers, octamers, and filamentous assemblies. To dissect the contributions of two oligomeric interface residues, L117 and W95, in the Sudan virus (SUDV) VP40 (sVP40), we created variants carrying alanine substitutions and assessed their structural and functional properties. sVP40 L117A failed to form dimers and was predominantly monomeric showing increased structural flexibility, reduced thermal stability together with loss of plasma membrane transport, budding activity, and the ability to regulate viral RNA synthesis. VP40 W95A preserved dimerization but also exhibited increased structural flexibility and reduced thermal stability. Functionally, sVP40 W95A more strongly inhibited viral RNA synthesis and markedly enhanced budding. However, in a transcription- and replication-competent virus-like particle (trVLP) assay, trVLPs produced with sVP40 W95A induced substantially reduced reporter activity in target cells, indicating impaired particle infectivity or functionality and suggesting possible defects in minigenome packaging, entry, or early post-entry steps. These results demonstrate that mutations at key oligomerization interfaces exert distinct structural and functional effects and highlight the requirement for precise oligomerization in coordinating sVP40s dual roles in genome regulation and virion release. By defining the contributions of L117 and W95, this study advances mechanistic understanding of sVP40 function and identifies processes that may serve as targets for antiviral intervention. ImportanceSudan virus (SUDV) causes regular outbreaks in Sub-Sahara Africa with unusually high lethality rates. However, in contrast to the more often occurring Zaire ebolavirus (EBOV), no monoclonal antibodies or vaccines are available and SUDV is generally understudied. The matrix protein VP40 is responsible for the downregulation of viral genome replication and transcription as well as budding. Here, we present structural and functional characterization of the SUDV VP40 interface residues L117 and W95 and show that while both amino acids are crucial for VP40s structural integrity, their functional effects are dramatically different ranging from complete abolishment to improving regulatory and budding activities.

microbiology↗

bioRxiv2025

Structural evolution of a fungal cell wall protein family for β-glucan-binding and cell separation

In fungi, the continuous biosynthesis and remodeling of the cell wall are crucial for growth, division, and development. A hallmark of fungal cell walls is their layered structure, which includes several gel-like carbohydrate polymers, such as {beta}-glucans, and a large number of associated cell wall proteins. The fungal-specific family of SUN domain proteins has been implicated in cell wall remodeling and cell separation, but detailed structure-based analyses revealing precise molecular functions have been lacking until now. In this study, we determined high-resolution crystal structures of the SUN domains from two paralogs of the SUN family in budding yeast. We find that their bilobal architecture consists of a sushi-like domain and an intimately associated thaumatin-like domain, which together form a highly conserved canyon fitted to accommodate both single- and triple-helical {beta}-glucan polymers. Within this canyon, we identify twelve conserved polar residues that are crucial for the function of SUN domains in mediating cell separation. We further demonstrate that SUN domains are functionally interchangeable between paralogs in budding yeast, as well as between orthologs from budding yeast and phylogenetically distant fission yeast or filamentous fungi. We conclude that the fungal SUN domain family represents a unique class of {beta}-1,3-glucan-binding proteins involved in cell wall remodeling and separation, whose successful evolution was enabled by the fusion of ancestral sushi- and thaumatin-like domains. ImportanceFungal cell walls are dynamic extracellular structures essential for growth and morphogenesis, making them prime targets for antifungal drugs and the host immune system. Although many protein families involved in the synthesis, crosslinking, and degradation of cell wall polymers are known, the molecular functions and structural evolution of most cell wall proteins remain poorly understood. Our in-depth structural, functional, and phylogenetic analysis of the fungal SUN domain protein family sheds light on a central question: how specific protein families have evolved structurally to enable dynamic cell wall remodeling during growth and division. Moreover, this work identifies precise structural targets within the fungal cell wall that could guide the development of novel diagnostics and therapeutics against life-threatening fungal infections.

microbiology↗

bioRxiv2025

Mechanistic insight into the oligomerization of Arabidopsis CRY1 and its inhibition by BIC1

Cryptochromes (CRY) convert blue light signals into biological responses, however, the molecular processes underlying their activation are not fully understood. In this study, we uncover the molecular mechanism underlying the blue-light activation of Arabidopsis CRY1 using time-resolved native mass spectrometry combined with kinetic modelling - an approach that allows us to monitor light-driven complex formation with temporal and molecular resolution. We show that CRY1 activation follows a defined, reversible assembly pathway in which monomers rapidly form dimers that then assemble into tetramers. A quantitative two-step model captures the dynamic interplay between light-driven assembly and thermal disassembly. Strikingly, ATP accelerates tetramer formation and stabilises oligomers by tuning the underlying photochemistry of the FAD chromophore. In contrast, the Blue-light Inhibitor of Cryptochromes 1 (BIC1) acts as a potent antagonist. We found that BIC1 binds to CRY1 even in the dark, with a significant increase in binding strength under blue-light conditions. BIC1 not only blocks CRY1 oligomerisation, but also actively dismantles pre-assembled tetramers. This disassembly process is light-independent and occurs regardless of CRY1s redox state. Together, these findings reveal a finely balanced regulatory system in which ATP and BIC1 act as opposing regulators to control CRY1 activation. This work provides a kinetic and mechanistic framework for reversible cryptochrome signalling and highlights how blue light responses can be precisely modulated at the molecular level.

plant biology↗

bioRxiv2023

Archaeal self-activating GPN-loop GTPases involve a lock-switch-rock mechanism for GTP hydrolysis

Three GPN-loop GTPases, GPN1-GPN3, are central to the maturation and trafficking of eukaryotic RNA polymerase II. This GTPase family is widely represented in archaea but typically occurs as single paralogs. Structural analysis of the GTP- and GDP-bound states of the Sulfolobus acidocaldarius GPN enzyme (SaGPN) showed that this central GPN-loop GTPase adopts two distinct quaternary structures. In the GTP-bound form the {gamma}-phosphate induces a tensed dimeric arrangement by interacting with the GPN region that is relaxed upon hydrolysis to GDP. Consequently, a rocking-like motion of the two protomers causes a major allosteric structural change towards the roof-like helices. Using a lock-switch-rock (LSR) mechanism, homo- and heterodimeric GPN-like GTPases are locked in the GTP-bound state and undergo large conformational changes upon GTP hydrolysis. A{Delta} saGPN strain of S. acidocaldarius was characterized by impaired motility and major changes in the proteome underscoring its functional relevance for S. acidocaldarius in vivo. Significance StatementGPN-loop GTPases have been found to be crucial for eukaryotic RNA polymerase II assembly and nuclear trafficking. Despite their ubiquitous occurrence in eukaryotes and archaea the mechanism by which these self-activating GTPases mediate their function is unknown. Our study on an archaeal representative from Sulfolobus acidocaldarius showed that these dimeric GTPases undergo large-scale conformational changes upon GTP hydrolysis, which can be summarized as a lock-switch-rock mechanism. The observed requirement of SaGPN for motility appears to be due to its large footprint on the archaeal proteome.

biochemistry↗

bioRxiv2022

Structural basis of bifunctionality of Sinorhizobium meliloti Clr, a cAMP and cGMP receptor protein

In bacteria, Crp-Fnr superfamily transcription factors are the most ubiquitous receptor proteins of 3,5-cyclic adenosine monophosphate (cAMP) and 3,5-cyclic guanosine monophosphate (cGMP). The prototypic Escherichia coli CAP protein represents the main CRP subclass and is known to bind cAMP and cGMP, but to mediate transcription activation only in its cAMP-bound state. In contrast, both cyclic nucleotides mediate transcription activation by CRP subclass G protein Clr of Sinorhizobium meliloti. We present crystal structures of apo-Clr, and Clr*cAMP and Clr*cGMP bound to the core motif of the palindromic Clr DNA binding site (CBS). We show that both cyclic nucleotides shift ternary Clr*cNMP*CBS-DNA complexes to almost identical active conformations. Unlike the situation known for the E. coli CAP*cNMP complex, in the Clr*cNMP complex, the nucleobases of cGMP and cAMP are in the syn- and anti-conformation, respectively, allowing a shift to the active conformations in both cases. Isothermal titration calorimetry measured similar affinities of cAMP and cGMP binding to Clr in presence of CBS core motif DNA (KDcNMP 16 M). However, different affinities were determined in absence of this DNA (KDcGMP 24 M; KDcGMP 6 M). Sequencing of Clr co-immunoprecipitated DNA as well as Electrophoretic Mobility Shift and promoter-probe assays expanded the list of experimentally proven Clr-regulated promoters and CBS. This comprehensive set of CBS features conserved nucleobases, which are in agreement with the sequence readout through interactions of Clr amino acid residues with these nucleobases, as revealed by the Clr*cNMP*CBS-DNA crystal structures. IMPORTANCECyclic 3,5-adenosine monophosphate (cAMP) and cyclic 3,5-guanosine monophosphate (cGMP) are both long known as important nucleotide second messenger in eukaryotes. This is also the case for cAMP in prokaryotes, whereas a signaling role for cGMP in this domain of life has been recognized only recently. Catabolite repressor proteins (CRPs) are the most ubiquitous bacterial cAMP receptor proteins. Escherichia coli CAP, the prototypic transcription regulator of the main CRP subclass, binds both cyclic mononucleotides, but only the CAP*cAMP complex promotes transcription activation. In contrast, CRP subclass G proteins studied so far, are activated by cGMP, or both by cAMP and cGMP. Here, we report a structural analysis of the bifunctional cAMP- and cGMP-activatable Clr from Sinorhizobium meliloti, how binding of cAMP and cGMP shifts Clr to its active conformation, and the structural basis of its DNA binding site specificity.

microbiology↗

bioRxiv2021

Interaction between BID and VDAC1 is required for mitochondrial demise and cell death in neurons

Mitochondrial damage is a key feature of regulated cell death in neurons. In particular, mitochondrial outer membrane permeabilization (MOMP) has been proposed as a starting point for mitochondrial demise upon cellular stress. Potential mechanisms for MOMP presented in the literature include membrane pore formation by Bcl2-family proteins such as BID and BAX, oligomerization of voltage-dependent anion channels (VDACs) and hetero-oligomer formation of these proteins. In our study, we demonstrate a direct interaction between the voltage-dependent anion channel VDAC1 and the pro-apoptotic protein BID in dying neurons both in vitro and in vivo. Binding of BID to VDAC1 affects anion conductance through VDAC1 and is associated with glutamate-induced cell death in cultured neurons and ischemic brain injury. In cultured neurons, reducing VDAC1 expression significantly attenuates BID-induced hallmarks of mitochondrial damage such as mitochondrial fission, declined mitochondrial respiration, increased ROS production, and mitochondrial membrane potential breakdown. Our data highlight a critical role for VDAC1 as a mitochondrial receptor for activated BID, thereby serving as a key decision point between life and death in neurons. One Sentence SummaryVDAC1 interacts with BID to mediate mitochondrial membrane permeabilization and neuronal cell death.

molecular biology↗

bioRxiv2021

The archaeal triphosphate tunnel metalloenzyme SaTTM defines structural determinants for the diverse activities in the CYTH protein family

CYTH is a large protein superfamily that is conserved in all three domains of life with its unique triphosphate tunnel metalloenzyme (TTM) fold. Besides phosphatase functions, e.g. as RNA triphosphatase, inorganic polyphosphatase or thiamine triphosphatase, some CYTH orthologs cyclize nucleotide triphosphates to 3,5-cyclic nucleotides. So far, archaeal CYTH proteins are annotated as adenylyl cyclases although experimental evidence is lacking. To address this gap, we characterized a CYTH ortholog, SaTTM, from the crenarchaeote Sulfolobus acidocaldarius. Our initial in silico studies suggested a close relationship between archaeal CYTH enzymes and class IV adenylyl cyclases compared to the other CYTH-subclasses, but biochemical data showed no cyclic nucleotide production. Instead, our structural and functional analyses show a classical TTM behavior. The Ca2+-inhibited Michaelis complex indicates a two-metal ion reaction mechanism analogous to other TTMs. Different co-crystal structures of SaTTM further reveal conformational dynamics in SaTTM, let us to assume feedback inhibition in TTMs due to tunnel closure in the product state. Combining our structural insights with sequence-similarity network based in silico analysis, we further set out a firm molecular basis for distinguishing CYTH orthologs with phosphatase activities from class IV adenylyl cyclases. Major highlights- CyaB-like class IV adenylyl cyclase homologs in archaea are triphosphatases. - The co-crystal structure of SaTTM in sulfate and triphosphate bound state revealed conformational transition of the TTM tunnel during catalysis. - Atomic insights into TTM inhibition by calcium and pyrophosphate. - In silico and structure-function analysis revealed the molecular determinant for functional diversification among CYTH proteins.

biochemistry↗